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animals and X-ray photographs were taken using a micro-CT scanner (Skyscan1071, Skyscan, Antwerp, Belgium). Samples were scanned with scanning direction perpendicular to the sagital aspect of bone.
High-resolution scanning, with an in-plane pixel size and thickness of 18 µm, was performed. The micro-CT scanners built-in software was used to make a 3-D reconstruction from the set of scans.
4.2.4 Histological Examination
Undecalcified sections were used for the histological examination. At 20 weeks after implantation, rabbit bone was fixed with a fixative solution 10% formaldehyde in 0.1 mol/L phosphate buffer in pH 7.4 for 1 day. Each specimen was dehydrated through a graded series of ethanol solutions and then embedded in acrylic resin. Sections of 150 µm in thickness were prepared and stained with hematoxylin and eosin.
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Figure 4-2 Micro-CT images of the operated portion of the rabbit femur (a) 4 weeks, (b) 12 weeks and (c) 20 weeks after implantation of TCP foams.
4.3.2 Micro-CT Images After Implantation of HAp Foam
Figure 4-3 shows the typical micro-CT images after implantation of HAp foam. After 4 weeks of implantation, still no dissolution of HAp foam was confirmed. After 12 weeks of implantation, no dissolution of HAp foam was confirmed. At this stage, newly formed bone was observed around the HAp foam. Moreover, bone formation inside the pores was
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observed as indicated by the arrows. After 20 weeks of implantation, bone defect was healed, keeping HAp foam as implanted.
Figure 4-3 Micro-CT images of the operated portion of the rabbit femur (a) 4 weeks, (b) 12 weeks and (c) 20 weeks after implantation of HAp foams.
4.3.3 Micro-CT Images After Implantation of Mg-βTCP Foam
Figure 4-4 shows the typical micro-CT images after implantation of Mg-βTCP foam. After 4 weeks of implantation, Mg-βTCP foam remained as implanted. After 12 weeks of implantation, Mg-βTCP foam still
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remained as implanted, but newly formed bone was observed. Moreover, bone formation inside the pores was observed as indicated by the arrows.
After 20 weeks of implantation, Mg-βTCP foam remained and bone defect was healed. At this stage, some Mg-βTCP foam were dissolved.
Figure 4-4 Micro-CT images of the operated portion of the rabbit femur (a) 4 weeks, (b) 12 weeks and (c) 20 weeks after implantation of Mg-βTCP foams.
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4.3.4 Micro-CT Images After Implantation of HT-βTCP Foam
Figure 4-5 shows the typical micro-CT images after implantation of HT-βTCP foam. After 4 weeks of implantation, although little HT-βTCP foam was dissolved, it was almost intact. After 12 weeks of implantation, some HT-βTCP foam remained and bone formation inside the pores was observed as indicated by the arrows. After 20 weeks of implantation, HT-βTCP foam was almost dissolved and bone defect was healed.
Figure 4-5 Micro-CT images of the operated portion of the rabbit femur (a) 4 weeks, (b) 12 weeks and (c) 20 weeks after implantation of HT-βTCP foams.
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4.3.5 Histological Appearances
Figure 4-6 shows the typical histological pictures of the foams when implanted in rabbit femur for 20 weeks. The newly formed bone after implantation of αTCP foam was restricted to the edge of the defects and most of the bone defect was occupied with non-osseous tissue. In contrast, new bone was formed and bone defect was healed with osseous tissue when treated with HAp foam, Mg-βTCP foam and HT-βTCP foam. HAp foam and Mg-βTCP foam surfaces were surrounded by bone tissue and strut of foams were fully integrated with newly formed bone. In contrast, residual strut of the foam could not be observed after implantation of HT-βTCP foam and the bone defect was filled with bone tissue similar to the normal femur bone.
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Figure 4-6 Typical histological appearances at 20 weeks after implantation of (a) αTCP foam, (b) HAp foam, (c) Mg-βTCP foam and (d) HT-βTCP foam. Sectioned samples were stained with hematoxylin and eosin.
Asterisks (*) represent the strut of the foam.
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4.4 Discussion
Results obtained in this study demonstrated clearly that both βTCP foams showed good osteoconductivity and bioresorbability. In other words, dissolution of the βTCP foam and bone formation occurred simultaneously in bone defect of rabbit. As stated in Chapter 1, bone formation and bioresorbability should be balanced for the ideal bone replacement. After 12 weeks of implantation, newly formed bone was observed in the pores of respective types of βTCP foam. Bone formation inside the pore was also observed in the case of HAp foam. HAp is known to show good osteoconductivity. Therefore, βTCP foams prepared in this study are considered to also have good osteoconductivity.
Bioresorbability may be a more difficult factor for bone replacement.
HAp is known to show almost no dissolution in bone. In the case of HAp foam as well, no bioresorbability was observed in bone defect of rabbit. In contrast, solubility of αTCP is high. Therefore αTCP powder has been used as a powder component for apatite cements. Although, specific surface area is significantly smaller in the case of αTCP foam, αTCP foam also showed too high bioresorbability as bone replacement in bone defect of rabbit. In other words, αTCP foam was dissolved completely 12 weeks after implantation without bone formation. Results of HAp foam and αTCP foam confirmed that balance of dissolution and bone formation is the key for ideal bone replacement.
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Fortunately, βTCP is known to have suitable bioresorbability as bone replacement. Therefore, βTCP foams prepared in this study was also expected to show proper bioresorbability. As expected, it was confirmed that replacement of βTCP foam to bone, or the dissolution of βTCP foam and new bone formation occurred simultaneously in rabbit bone for both Mg-βTCP foam and HT-βTCP foam (Figure 4-4, 4-5). It should be noted that bioresorbability of Mg-βTCP foam and HT-βTCP foam were different.
After 12 weeks, no indication of bioresorbability was observed in the case of Mg-βTCP foam, whereas some bioresorption was observed in the case of HT-βTCP foam. After 20 weeks of implantation, HT-βTCP foam was almost dissolved and replaced to new bone, whereas some structure of Mg-βTCP foam still remained in rabbit bone. It was reported that the solubility of βTCP decreases with the Mg incorporation into the βTCP structure, since substitution of Mg leads to stabilization of original βTCP structure [59, 60]. Similarly, bioresorbability of Mg-βTCP foam was thought to be decreased in bone defect of rabbit. In other words, bioresorbability of βTCP foam could be controlled by the added Mg into the βTCP structure.
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4.5 Conclusion
The results of this in vivo study demonstrated that βTCP foams showed good osteoconductivity and proper bioresorbability. The bone defect was healed and HT-βTCP foam was replaced to new bone after 20 weeks of implantation. The bone defect was also healed after 20 weeks implantation in the case of Mg-βTCP foam, although some Mg-βTCP foam still remained undissolved in the bone at this stage. These TCP foams are promising candidates for the ideal bone replacement.
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CHAPTER 5 Summary
βTCP foam is thought to be an ideal bone replacement because (1) it would be replaced to new bone due to balanced bioresorbability and bone formation and (2) it has fully interconnected porous structure that allows tissue ingrowth and nourishment supply to bone cells. However, TCP foam has not been fabricated using the polyurethane foam replica method up to date due to insufficient sintering reaction when sintered below -
transition temperature. In this study, new methods was proposed to fabricated βTCP foam. One is the use of β phase stabilizer and the other is the phase transformation from αTCP to βTCP based on heat treatment.
In Chapter 2, TCP foam was found to be fabricated by employing MgO as TCP stabilizer. 3 mol% or larger amount of Mg was the key to stabilize TCP phase when sintered at 1,500C. The compressive strength of the TCP foam fabricated using MgO stabilizer was similar regardless of the amount of added Mg.
In Chapter 3, pure TCP foam was prepared based on phase transformation of sintered TCP foam by heat treatment below α-β transition temperature. The heat treatment at 800°C to 1,000°C resulted in complete phase transformation from αTCP to TCP. Heat treatment at 1,000ºC for 300 hours resulted in highest compressive strength or the same
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compressive strength with that of TCP foam.
In Chapter 4, osteoconductivity and bioresorbability was evaluated using experimental animals, rabbits. Micro-CT scan analysis and histological analysis demonstrated that dissolution and replacement to newly formed bone occurred simultaneously in rabbit bone for both Mg-βTCP foam and βTCP foam fabricated by heat treatment (HT-βTCP foam). Especially, HT-βTCP foam showed proper osteoconductivity and bioresorbability in the condition employed in this animal study. HT-βTCP foam replaced to new bone after 20 weeks of implantation. Mg-βTCP foam also showed bioresorbability and thus, the bone defect was healed after 20 weeks implantation. However some Mg-βTCP foam still remained even at 20 weeks. In other words, Mg stabilizer is also useful for the regulation of bioresorbability.
In conclusion, the results of the present study demonstrated that TCP foam with fully interconnected porous structure can be fabricated in two methods. One is using Mg as β phase stabilizer, and the other is heat treatment of the αTCP foam. The βTCP foams thus prepared could be ideal bone replacements which can be replaced to new bone.
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Acknowledgements
I am deeply grateful to Professor Kunio Ishikawa who provided me constructive direction for carrying out this research. He also gave several good opportunities for me to learn.
I am also deeply grateful to Professor Seiji Nakamura who welcomed and accepted me in Department of Oral and Maxillofacial Oncology.
A heartfelt thanks to Associate Professor Kanji Tsuru who taught me concrete experimental technique and fruitful discussion in my daily research life.
The suggestions from Professor Shigeki Matsuya and Assistant Professor Michito Maruta of Fukuoka Dental College were absolutely necessary for my research study.
To Dr. Fumikazu Daitou, Dr. Mami Miyazaki and Dr. Kanako Matsubara who are alumni of our laboratory, for giving me invaluable supports.
Special thanks to members in our laboratory, Dr. Melvin Munar, Dr.
Girlie Munar, Dr. Giichiro Kawachi and Dr. Syunsuke Nomura. I would like to thank each of you and I am very glad to do research with you. And I would like to thank my junior collegues.
In addition, I am very thankful to all the doctors in Department of Oral